Target enrichment of metazoan USCO genes of Pachypus sardiniensis specimen 836901
Genomic DNA was extracted using Qiagen Blood & Tissue Kits. 100 ng genomic DNA (if available) was sheared with a Bioruptor PICO sonicator to gain approximately 350 bp long DNA fragments. End-repair and A-tailing with Agilent SureSelectXT2 Reagent Kit, NEBNext Quick Ligation Module, NEBNext Multiplex Oligos for Illumina (Dual Index Set1) and NEBNext Q5 HotStart HiFi PCR Master Mix were used for pre-capture adaptor ligation and dual-indexing amplification. PCR-program: ten or twelve or 16 cycles (98C/10s65C/75s), the number of cycles was dependent on concentrations after a-tailing concentration measurement. Between these steps, samples were purified with AMPure XP beads. After amplification, equal DNA amounts of eight samples a time were pooled. SureSelect XT2 Pre-Capture ILM Module Box 2 Kit was used for the following hybridization steps. After dsDNA was denatured for 5 min at 95 C in a thermal cycler, hybridization took place at 65 C for approximately 48 h. The library of hybridized baits and DNA fragments was captured with Dynabeads MyOne Streptavidin T1 beads. The captured libraries were amplified in twelve cycles (for bad samples 14 cycles) using the SureSelect XT2 primer mix and Herculase II PCR Master Mix and purified with AMPure XP beads. Libraries were sequenced on an Illumina Nextseq 500 platform with 260 million reads.